Instrumental Analysis
Control
Lecture No. (07)
0
Introduction to Electrophoresis
Electrophoresis: Types, Applications, Advantages, Disadvantages
• Electrophoresis is a method of separating molecules based on their ability to move in an
electric field. Electrophoresis has become the most extensively used method for analyzing
biomolecules in biochemistry or molecular biology, including genetic components such as
DNA or RNA, proteins, and polysaccharides.
Principle of electrophoresis
• When a potential difference is introduced, molecules with different overall charges start to
segregate due to differences in electrophoretic mobility.
• Even molecules with equal charges will begin to split if their molecular sizes differ because
they would encounter distinct frictional forces.
• As a result, certain forms of electrophoresis rely almost entirely on the various charges on
molecules for separation, whilst others rely on differences in the size (molecular size) of
molecules.
• The migration and separation of charged particles (ions) under the influence of an electric
field are referred to as electrophoresis.
• An electrophoretic system consists of two electrodes of opposite charge (anode, cathode)
linked by a conducting substance known as an electrolyte.
• The separation effect on ionic particles is caused by changes in velocity (v), which is the
product of particle mobility (m) and field strength (E):
V = ME
An ionic particle’s mobility (m), which is constant under specific electrophoretic circumstances, is
dictated by the particle’s size, shape, charge, and temperature during separation.
The rate of movement of charged molecules is affected by the following factors:
(a) The electric field’s strength, size, and shape.
1
(b) The sample’s relative hydrophobicity.
(c) The buffer’s ionic strength and temperature.
(d) The biomolecule’s molecular size.
(e) The biomolecule’s net charge density.
(f) The biomolecule’s shape.
Types of electrophoresis
• Electrophoresis is the movement of charged molecules through liquids in an electric field.
Electrophoresis is frequently characterized based on the presence or absence of a solid
support medium or matrix through which the charged molecules in the electrophoretic
solution migrate.
• Electrophoresis can be categorized into two types based on the presence or absence of
supporting media:
free electrophoresis
zone electrophoresis.
Free electrophoresis
• Aqueous buffers are used in solution electrophoresis systems. Such systems may experience
sample mixing mainly as a result of charged molecule diffusion, resulting in a loss of resolution
during the sample application, separation, and removal steps. As a result, solution
electrophoresis systems must employ some method of aqueous solution stabilization in the
electrophoresis cell.
To minimize diffusional mixing of the materials being separated during electrophoresis, soluble-gradient
electrophoresis systems, for example, use variable densities of a non-ionic solute (e.g., sucrose or
glycerol). Despite these improvements, solution electrophoresis devices only have limited uses, typically
where preparative scale electrophoretic separation is required.
Zone electrophoresis
• Zone electrophoresis is similar to moving boundary electrophoresis in that it uses a
homogeneous buffer solution to separate proteins.
• This format usually employs a support medium or matrix to decrease convection current and
avoid uncontrolled sample diffusion.
• In most cases, the matrix has an additional sieving action that affects electrophoretic separation.
Samples for ZE separation are enclosed in an electrophoretic solution buffer and separated in
the matrix for a predetermined amount of time.
2
• When an electric current (q) is applied to the sample, it moves at different speeds depending
on its mass and charge. After the separation operation is completed, components of the sample
with comparable qualities are divided into a separate zone.
There are mainly four types of zone electrophoresis:
(a) paper electrophoresis,
(b) cellulose acetate electrophoresis,
(c) capillary electrophoresis,
(d) gel electrophoresis.
3
Paper electrophoresis
• In this type of electrophoresis, a filter paper (similar to chromatography paper) with a low
adsorption capacity and homogeneous pore size serves as the supporting medium for
sample separation under the effect of an applied electric field.
Cellulose acetate electrophoresis
• It is a modified form of paper electrophoresis, which Kohn invented in 1958.
Bacteriological acetate membrane filters are used instead of ordinary chromatography
paper in this sort of electrophoresis.
• The advantages of cellulose acetate strip over chromatography paper are as follows:
a. The cellulose acetate strips are chemically pure and devoid of lignin and
hemicelluloses, and they serve as barriers in the free moment of big molecules in
general.
b. Due to the low content of glucose cellulose acetate strips, it is ideal for
polysaccharide electrophoresis.
c. Since cellulose acetate is not hydrophilic, it holds very little buffer, allowing for faster
resolution.
Gel electrophoresis
• Gel electrophoresis is a technique for separating DNA fragments (or other macromolecules like
RNA and proteins) depending on size and charge.
• Electrophoresis is the process of passing a current across a gel containing the molecules of
4
interest.
• The molecules will go through the gel in different directions or at different speeds depending on
their size and charge, allowing them to be segregated from one another.
Agarose gel electrophoresis
• Agarose gel is used as the supporting medium in this form of electrophoresis. This is used for
nucleic acid electrophoresis, such as DNA and RNA.
• When a potential difference is applied across the electrodes of an agarose gel-filled horizontal
electrophoretic tank and biomolecules are loaded, they are separated based on their
molecular size and move to their respective electrodes. The agarose gel works as a sieve in
this case.
• In the same way that large particles remain above a sieve while particles smaller than the pore
size passes through it, larger and bulky molecules remain behind while smaller molecules
flow faster and more swiftly toward their respective electrodes.
• Smaller proteins migrate quicker due to less resistance from the gel matrix when separated by
electrophoresis across a gel matrix. The structure and charge of the proteins also affect the
rate of migration across the gel matrix.
SDS-PAGE electrophoresis
• The use of sodium dodecyl sulfate (SDS, also known as sodium lauryl sulfate) and
polyacrylamide gel in SDS-PAGE greatly reduces the influence of structure and charge, and
proteins are separated based on polypeptide chain length.
• SDS is a detergent that binds to the protein backbone at a constant molar ratio and has a
significant protein-denaturing effect.
• Proteins unfold into linear chains with negative charge proportionate to polypeptide chain
length in the presence of SDS and a reducing agent that cleaves disulfide bonds required for
optimal folding.
Capillary electrophoresis
• The narrow bore tube’s capillarity is used to separate the samples depending on their size:
charge ratio.
• In comparison to established separation techniques such as agarose gel electrophoresis or
SDS-PAGE, capillary electrophoresis (CE) is a relatively new separation technique.
• It has exceptional qualities that make it both competitive and a good option. The capacity to
separate charged and non-charged molecules is one of the key advantages of capillary
5
electrophoresis over other separation techniques.
• CE separates analyte ions in an electrolyte solution (background electrolyte) within a tiny fused
silica capillary.
Affinity electrophoresis
• Affinity Electrophoresis is a type of electrophoresis that separates a biomolecule that interacts
with and binds to another molecule for which it has an affinity.
• It makes use of the fact that when a biomolecule binds to another molecule, its electrical
mobility changes and this change in electrical mobility is recorded in the electrophoretic
pattern.
Factors affecting electrophoresis
Sample
• The migration rate of the sample being separated is affected by its charge, size, and shape. A net
increase in the charge accelerates migration. The rate of migration is determined by molecule
size (inversely proportional) and sample shape.
Buffer solution
• Buffer influences compound migration rate and stabilizes the pH of the supporting medium.
• It has been discovered that zwitterionic buffers may resist continuous electrolysis far better than
standard buffers, particularly in capillary zone electrophoresis.
Frictional force
• A frictional force also slows the mobility of this charged molecule. This frictional force is a
measure of the molecule’s hydrodynamic size, shape, the pore size of the medium in which the
electrophoresis is taking place, and the viscosity of the buffer.
Applied voltage
• The voltage applied influences the travel time of the molecules being separated. The higher
the voltage, the faster the DNA will flow through the gel. However, excessive voltages may
melt the gel or produce smearing or distortion of DNA bands.
Supporting media
• The rate of compound migration is affected by the type of supporting media used. It is always
preferable to use inert media.
• Adsorption, molecular sieving, and electro- osmosis processes may occur in the medium,
affecting the electrophoretic rate.
6
• The sample moves like a comet rather than a band as a result of tailing caused by
adsorption. This decreases both the rate and the resolution of the separation.
Electroendosmosis
• Electroendosmosis (also known as electro-osmotic flow) is an important factor that might alter
electrophoretic separation.
• The presence of charged groups on the surface of the support medium causes this
behavior. Paper, for example, includes some carboxyl groups, agarose contains sulfate
groups depending on the purity grade, and the surface of glass walls used in capillary
electrophoresis contains silanol (Si-OH) groups.
• At the suitable pH, these groups will ionize, resulting in charged sites. These charges are
responsible for electroendosmosis. In the case of capillary electrophoresis, the ionized silanol
groups form an electrical double layer, or a charge separation area, at the capillary
wall/electrolytic contact.
• When a voltage is given to the electrolyte near the capillary walls, cations in the electrolyte
migrate toward the cathode, dragging the electrolyte solution with them. This results in a net
electroosmotic flow towards the cathode.
Applications of electrophoresis
DNA analysis
One of the most common uses for electrophoresis is DNA analysis. Researchers can divide DNA
into segments using an electrical charge and maintain the molecules in place once the charge is
withdrawn using the gel as a medium. This enables researchers to examine molecules at high
resolutions, making full analysis of DNA architecture much easier.
Explosives chemical and residue analysis
Capillary electrophoresis is employed in the trace analysis of organic and inorganic gunshot residues
and explosives. Organic gunpowder additives such as ethylcentralite, diphenylamine, and
nitroglycerin can be examined using Miceller Electro kinetic capillary chromatography.
Protein detection
Immunoelectrophoresis is a sort of electrophoresis that is commonly used to examine the
existence of various types of proteins and how they react chemically in different settings. When
abnormal protein molecules form, they become stimulated by several medical problems such as
multiple sclerosis, kidney failure, and even various types of cancer. The irregular proteins are
detected using electrophoresis on urine or blood samples and the results are constantly monitored
for any variationsfrom typical protein shapes and levels. Immunoelectrophoresis is also used to
identify particular proteins known as immunoglobulins.
Analysis of drug abuse:
7
One-of-a-kind organic samples, for example, tissue, hair, nail, and body fluids, are often used in the
detection of illicit tablets. Capillary electrophoresis is used to detect drugs.
Test for antibodies
When it comes to antibiotic testing, electrophoresis serves several important roles.
The testing of antibiotics to ensure their purity is one of the most prevalent applications of the
electrophoresis technique in this industry. Electrophoresis is utilized in a solution containing the
antibiotic to be tested on a paper strip. This strip is impregnated with a capillary or antibiotic that
contains the medicine.
Electrophoresis is also used to determine the potency of the antibiotic, which is critical in providing the
correct dosages. In addition, the antibiotic research and genetic testing fields share a common ground.
As a result, electrophoresis aids in the identification of genes that signal resistance to a specific type
of antibiotic.
Vaccine testing
Electrophoresis has been critical in the creation of modern vaccines. it is used to test vaccine quality
and concentration. To determine the best potential form of a single vaccine, researchers utilize
electrophoresis to evaluate different varieties of vaccines with varying quantities and types of
antibodies.
Advantages of electrophoresis
It has a high efficiency of separation.
It provides sample analysis in a short period of time.
It produces fewer waste products.
It is a simple strategy to use.
The experiment can be performed with a small amount of sample.
Disadvantages of electrophoresis
During electrophoresis, gels can melt, the buffer can run out, and various genetic materials can run in
unanticipated manners.
Heat is dissipated by the capillary tube’s narrow diameter, resulting in greater diffusion. As a result, the
resolution is not always accurate.
References
1. Ferrier D. R. (2017). Lippincott illustrated reviews: biochemistry (Seventh). Wolters
Kluwer.
2. https://scienceinfo.com/electrophoresis-types-applications-advantage/
